In recent years, the bio-imaging technique for imaging a distribution of biological substances (such as proteins or metabolites) in a section of biological tissue has been used in the fields of clinical diagnoses or drug discoveries. In particular, this technique is widely used for an investigation of a distribution of biological substances, in which a section sample prepared by an appropriate pretreatment of a paraffin-embedded tissue section is scanned by a MALDI (matrix-assisted laser desorption ionization) mass spectrometer, and the thereby measured masses of biological substances in the section sample are comprehensively analyzed to determine the distribution of the biological substances.
A pretreating method described in Non-Patent Documents 1, 2, 3 and 4 has conventionally been used to make a paraffin-embedded tissue section available as a section sample for a mass spectrometry. This conventional pretreatment method is hereinafter described by means of the flowchart shown in FIG. 2.
Initially, paraffin is removed from the paraffin-embedded tissue section, after which a hydration treatment is performed to obtain a deparaffinized tissue section (S1). Inside the molecules of the proteins contained in the deparaffinized tissue section thus obtained, bonding of single amino acid residues or a formation of methylene crosslink by a hydroxyl group, in particular, a strong methylene crosslink via a primary amino or phenyl radical, have occurred. To dissociate such bonds or crosslinks (both are referred to as “crosslinks” hereinafter), the tissue section is subjected to a heat treatment in a retrieval treatment solution (S2: this treatment is hereinafter called the “retrieval treatment”). Subsequently, the retrieval treatment solution which has adhered to the deparaffinized tissue section during the retrieval treatment is washed off (S3). After the washing, the deparaffinized tissue section is treated with a digestive enzyme, such as trypsin, to hydrolyze proteins contained in the tissue section into peptides (S4). Lastly, after the enzymatic treatment, a matrix solution is dropped onto the deparaffinized tissue section to deposit the matrix on it (S5). The section sample thus prepared is subjected to the MALDI mass spectrometry.
The retrieval treatment in the previously described conventional pretreatment method is not only indispensable for restoring two-dimensional and three-dimensional structures of the protein molecules which have been damaged due to the crosslinking reaction caused by the formalin fixation treatment, but also for facilitating an approach of the digestive enzyme to protein molecules in the subsequent enzymatic treatment. In one conventional retrieval treatment, the deparaffinized tissue section is immersed in a retrieval treatment solution containing a surfactant, and a heat treatment (in a hot bath, a microwave oven or an autoclave) is performed on the immersed sample for a predetermined period of time.